Method for the treatment of apolipoprotein E related diseases

ABSTRACT

Methods for treating diseases associated with toxicity of Apolipoprotein E (“apoE”). Specifically, the present invention relates to new methods for treating a mammal having a condition associated with toxicity of apolipoprotein E cleavage fragments containing residues 130-169, comprising administering to said mammal a pharmacologically effective amount of compound or a pharmaceutically acceptable sale, derivative or fragment thereof to interfere with the receptor-binding site associated with residues 130-169 of the apolipoprotein E molecule in said mammal.

RELATED APPLICATION

[0001] This is a continuation-in-part of U.S. Ser. No. 09/214,742, filedJan. 6, 1999, which claims priority from international applicationPCT/US97/11836, filed Jul. 8, 1997, which is a continuation-in-part ofU.S. Ser. No. 60/021,405, filed Jul. 9, 1996, all hereby expresslyincorporated by reference.

SPONSORSHIP

[0002] The U.S. Government has a paid-up license in this invention andthe right in limited circumstances to require the patent owner tolicense others on reasonable terms as provided for by the terms of GrantNo. RO1 HL27333, NS31410 and R43 NS37986-01 awarded by the NationalInstitutes of Health.

BACKGROUND OF THE INVENTION

[0003] The present invention relates generally to a method for treatingdiseases associated with toxicity of Apolipoprotein E (“apoE”).Specifically, the present invention is a new method for treating amammal having a condition associated with toxicity of apolipoprotein Eand/or apolipoprotein E cleavage fragments containing residues 130-169,comprising administering to said mammal a pharmacologically effectiveamount of a compound or a pharmaceutically acceptable salt, derivativeor fragment thereof to interfere with the toxicity mechanism associatedwith residues 130-169 of the apolipoprotein E molecule in said mammal.

[0004] Alzheimer's disease is the most common form of both senile andpre-senile dementia in the world and is recognized clinically asrelentlessly progressive dementia that presents with increasing loss ofintellectual function including memory and disturbances in speech(Merritt, A Textbook of Neurology,. 6th edition, pp. 484-489 Lea &Febiger, Philadelphia (1979)). The disease itself usually has a slow andinsidious progress that affects both sexes equally, worldwide. It beginswith mildly inappropriate behavior, uncritical statements, irritability,a tendency towards grandiosity, euphoria and deteriorating performanceat work; it progresses through deterioration in operational judgment,loss of insight, depression and loss of recent memory; it ends in severedisorientation and confusion, apraxia of gait, generalized rigidity andincontinence (Gilroy & Meyer, Medical Neurology, pp. 175-179 MacMillanPublishing Co. (1979)) Alzheimer's disease afflicts an estimated fourmillion human beings in the United States alone at a cost of 100 billiondollars a year (Schumock, G. T., J. Health Syst. Pharm. 55(52):17-21(1998); Hay & Ernst, Am. J. Public Health, 77:1169-1175 (1987)). It isfound in 10% of the population over the age of 65 and 47% of thepopulation over the age of 85 (Forsyth, E., Phys. Ther. 78:1325-1331(1998); Evans et al., JAMA 262:2551-2556 (1989)). In addition, thedisease is found at much lower levels in the younger age groups, usuallybeginning at about 30 years of age and even rarely in late childhood(Adams & Victor, Principles of Neurology, pp. 401-407 (1977)).

[0005] The etiology of Alzheimer's disease is unknown. Evidence for agenetic contribution comes from several important observations such asthe familial incidence, pedigree analysis, monozygotic and dizygotictwin studies and the association of the disease with Down's syndrome(for review see Baraitser, The Genetics of Neurological Disorders, 2ndedition, pp. 85-88 (1990)). Nevertheless, this evidence is far fromdefinitive and it is clear that one or more other factors are alsorequired.

[0006] In recent years, research has suggested that apolipoprotein E(“apoE”) plays a potential role in the pathogenesis of Alzheimer'sdisease. Apolipoprotein E performs various functions as a proteinconstituent of plasma lipoproteins, including its role in cholesterolmetabolism. It was first identified as a constituent ofliver-synthesized very low density lipoproteins (“VLDL”) which activityis the transport of triglycerides from the liver to peripheral tissues.ApoE is instrumental in lipoprotein metabolism in several ways (Mahley,et al., J. Lipid Res. 25:1277-1294 (1984)). It is a recognition site forseveral cellular lipoprotein receptors, including hepatocyte receptorsfor chylomicron and VLDL remnants (Hui, et al., J. Biol. Chem.,259:860-869 (1984); Shelburne, et al., J. Clin. Invest., 65:652-658(1980)).

[0007] Apo E-enriched lipoproteins have also been described to have afunction in the immune system by inhibiting mitogen- orantigen-stimulated lymphocyte proliferation in vitro and in vivo. In theovary, apo E inhibits androgen production by LH-stimulated culturedtheca and interstitial cells (Dyer, et al., J. Biol. Chem., 263:10965(1988)).

[0008] Further substantiation that apo E and apo B-containinglipoproteins are important regulators of lymphocyte function has comefrom studies of the inhibitory properties of fetal cord blood plasmalipoproteins (Curtiss, et al., J. Immunol., 133:1379 (1984)). In thesestudies a direct correlation between apo E and inhibition wasestablished.

[0009] There are three major isoforms of ApoE, referred to as ApoE2,ApoE3 and ApoE4 which are products of three alleles at a single genelocus. Three homozygous phenotypes (Apo-E2/2, E3/3, and E4/4) and threeheterozygous phenotypes (ApoE3/2, E4/3 and E4/2) arise from theexpression of any two of the three alleles. The most common phenotype isApoE3/3 and the most common allele is E3. See Mahley, R. W., Science240:622-630 (1988).

[0010] The amino acid sequences of the three types differ only slightly.ApoE4 differs from ApoE3 in that in ApoE4 arginine is substituted forthe normally occurring cysteine at amino acid residue 112. The mostcommon form of ApoE2 differs from ApoE3 at residue 158, where cysteineis substituted for the normally occurring arginine. See Mahley, Science,supra. ApoE phenotypes and genotypes are well described and known in theart as described above. The established nomenclature system as well asthe phenotypes and genotypes for ApoE, are described in, for example,Zannis, et al., J. Lipid. Res. 23:911 et seq. (1982), which isincorporated by reference herein.

[0011] Subjects with the ApoE4/4 genotype are as much as eight times aslikely to be affected by Alzheimer's disease as subjects with theApoE2/3 or ApoE3/3 genotypes. Further, the average age of onset ofAlzheimer's disease and the average age of survival is lower for thosehaving one ApoE4 allele, and lowest for those having two ApoE4 alleles(U.S. Pat. No. 5,508,167). Thus, a subject's prognosis for Alzheimer'sdisease is more likely to be negative if the subject has an ApoE4 alleleand most negative if the subject has more than one ApoE4 allele. Thenegative prognosis can be viewed in terms of increased likelihood ofdeveloping the disease, or of earlier age of onset. Other ApoE-linkeddiseases include type III Hyperlipidemia and atherosclerosis. Otherevidence indicates that polymorphisms in the apoE promoter are alsoassociated with increased risk of AD (Lambert et al., Human Mol. Gen.7:533 (1998); Lambert et al., Human Mol. Gen. 7:1511 (1998)).

[0012] Studies have shown that apoE fragments ranging from 5 to 22 kDaare present in the post-mortem cerebral spinal fluid from both controlpatients and patients with AD. The only major band immunoprecipitated bya monoclonal antibody that recognizes the putative toxic domain runswith an apparent molecular weight of about 22 kDa. This fragment likelycorresponds to the major apoE thrombin cleavage product, which has beenshown to be protease-resistant. Weisgraber, et al., J. Biol. Chem.258:12348-54 (1983).

[0013] Amino acids 130-169 in human apoE encompass an immunoregulatorydomain with both cytostatic and cytotoxic activities againstinterleukin-2-dependent T cells. This finding is consistent with resultsof previous studies (Cardin, et al., 1988; Dyer, et al., 1991) thatimplicated residues 141-155 in apoE's antiproliferative effect on naivemitogen-activated T cells. The similar potencies of E130-149 andE130-155 indicate that the cytostatic domain is located within residues130-149. However, a longer peptide representing residues 130-169 anddimeric peptides of amino acids 141-155 also have potent cytotoxicactivity. These results indicate that the positively charged,leucine-rich sequence, corresponding to amino acids 141-149(Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu; referred to as LRKLRKRLL in singleletter amino acid shorthand) in the mature protein which represents theoverlap between the functional peptides identified, is responsible forboth the cytostatic and cytotoxic effect (Clay et al., Biochemistry34:11142-51 (1995)). When tested against primary neurons in culture,these peptides were also found to elicit degeneration of neurites andneuronal cell death.

[0014] Purified apoE, derived from transfected HEK cells, subjected tothrombin cleavage and separated using gel filtration to collect the 22kD fragment, yields enhanced toxicity when tested against primaryneurons in culture. The 22 kD fragments purified from the E4 isoform aremore toxic than E3-derived fragments. The putative toxic site is closelyassociated with one of two well-characterized apoE heparin bindingdomains associated with residues 141-147 of apoE.

[0015] The density of four positively charged amino acid residues in the141-149 domain clearly make a significant contribution to apoE peptidetoxicity. Consistent with this conclusion is the ablation ofpeptide-mediated toxicity by the polyanionic glycosaminoglycans (“GAG”)heparin, heparan sulfate and chondroitin sulfate. However, GAG-bindingcapacity does not, in itself, account for bioactivity since peptideE211-243, which contains a second heparin-binding site but lacks the141-149 sequence, is inactive. Furthermore, not all GAGs show inhibitionof the toxicity.

[0016] While there has been considerable research into the mechanismsunderlying Alzheimer's disease, there continues to be an ongoing needfor new ways to investigate and combat this disorder and other diseasesin which ApoE has been implicated.

[0017] U.S. Pat. No. 5,795,860 discloses protein-specificglycosaminoglycan sequences and methods of use of such sequences inblocking the action of glycan-binding proteins.

[0018] U.S. Pat. No. 4,727,063 discloses low molecular weight heparinshaving a sulfation degree of at least 2.5 and a molecular weight rangingfrom 2000 to 9000, prepared by depolymerization and sulfation with amixture of sulfuric and chlorosulfonic acid. None has a sulfation degreeup to 3.5.

[0019] U.S. Pat. No. 3,454,560 discloses a process for thedepolymerization and sulfation of chondroitin sulfate by means ofsulfuric acid at a concentration not lower than 85% w/w. The sulfuricacid can contain another sulfating agent, such as sulfuric anhydride orchlorosulfonic acid, but the same document specifies that, evenoperating in said ambient, only sulfuric acid participates in thesulfation reaction.

[0020] U.S. Pat. No. 5,508,167, Roses et al., issued Apr. 16, 1996,discloses methods of diagnosing or prognosing Alzheimer's disease in asubject. The methods involve directly or indirectly detecting thepresence or absence of an apolipoprotein E type (ApoE4) isoform or DNA,encoding ApoE4 in the subject. The presence of ApoE4 indicates thesubject is afflicted with Alzheimer's disease or at risk of developingAlzheimer's disease. A novel immunochemical assay for detecting thepresence or absence of the Apolipoprotein E (ApoE) E4 allele in asubject is also disclosed.

[0021] U.S. Pat. No. 5,384,398, Lormeau et al., issued Jan. 24, 1995,discloses new high molecular mass N,O-sulphated heparosans consisting ofchains or of a mixture of chains having a molecular mass of between1.5×10⁴ and 4.0×10⁶D, characterized by a repeating disaccharidestructure.

[0022] U.S. Pat. No. 5,164,295, Kisilevsky et al., issued Nov. 17, 1992,discloses a method of identifying compounds which impair and/or preventinitiation and/or progression of amyloid deposition, such compoundsbeing useful as therapeutics for treating amyloidosis andamyloid-related disorders.

[0023] U.S. Pat. No. 4,956,347, Ban et al., issued Sep. 11, 1990,relates to the use of ATEROID, a mixture of “sulfomucopolysaccharides”comprising heparin, heparan sulfate-like substance, dermatan sulfate,and chondroitin sulfate A and C, for the treatment of patients sufferingfrom Alzheimer's-type senile dementia. ATEROID is defined in the U.S.Pat. No. 3,000,787, Bianchini, issued Sep. 19, 1961, as a heparinoidanti-cholesterolemic factor. ATEROID, which is in some aspects similarto heparin, has essentially no anti-coagulant effect. The patentdiscloses that ATEROID can be extracted from the small intestine andparticularly from the duodenum of mammals, by means of methods suitablefor the isolation of aminopolysaccharidic or glycoproteic compounds.

[0024] Snow, A. D., et al., American Journal of Pathology, Vol 133, No.3, December 1988, disclose the presence of heparan sulfate proteoglycan(HSPG) in neuritic plaques associated with Alzheimer's disease. HSPG wasdetected in the amyloid fibrils present in neuritic plaques in thebrains of Alzheimer's patients using antibodies against the protein coreof HSPG. Additionally, HSPG was shown to be present in primitiveplaques. It is suggested that the accumulation of HSPG in plaques takesplace during early stages of plaque development.

[0025] Snow, A. D., and Kisilevsky, R., Laboratory Investigation, Vol.53, No. 1, pp. 37-44 (1985), report the temporal relationship betweenglycosaminoglycan (GAG) accumulation and amyloid deposition duringexperimental amyloidosis. Using models which facilitate induction ofamyloidosis, it was disclosed that amyloid-associated GAGs appear in thetissues together with the AA amyloid protein independent of the tissuetype. It is suggested that the appearance of GAG in the inflammatoryamyloidosis condition appears to be part of the process involved in thedeposition of the AA protein.

[0026] Margolis, R. U., and Margolis, R. K., Neurobiology of Aging, Vol.10, pp. 500-502 (1989) disclose various properties of nervous tissueproteoglycans with respect to their proposed relation to amyloidbeta-protein in Alzheimer's disease-related amyloidosis. It is pointedout on page 501, column 1, lines 4 to 8 that the role of proteoglycansin Alzheimer's disease amyloidosis is only circumstantial and the roleof proteoglycans in it is unclear. At page 502, column 2, lines 3 to 6,it is disclosed that due to the absence of firm evidence specificallylinking proteoglycans to pathogenesis of Alzheimer's disease, it ispremature to speculate on the relationship of proteoglycans to amyloidin degenerative processes.

[0027] Caputo, C. B., Neurobiology of Aging, Vol. 10, pp. 503-504 (1989)refers to the significance of binding of proteoglycans to amyloid. It isdisclosed that co-localization of proteoglycans with amyloids indicatesthat they are binding but the consequence of such binding is unknown.The question is asked whether proteoglycans bind inadvertently toamyloid or whether the proteoglycans in binding to amyloid or itsprecursors lead to the formation of beta-pleated sheet conformation orthe stabilization of such a conformation. It is suggested that in vitrostudies be performed to determine whether Alzheimer amyloid precursorbinds to proteoglycans. On page 503, column 1, last paragraph, thepossibility that amyloid protein binds well to proteoglycans is raised.However, evidence is referred to which indicates otherwise.

SUMMARY OF THE INVENTION

[0028] The present invention relates generally to a method for treatingdiseases associated with toxicity of Apolipoprotein E (“apoE”).Specifically, the present invention is a new method for treating amammal having a condition associated with toxicity of wholeapolipoprotein E or apoE cleavage fragments containing residues 130-169,comprising administering to said mammal a pharmacologically effectiveamount of a compound or a pharmaceutically acceptable salt, derivativeor fragment thereof which interferes with the receptor-binding site ortoxicity associated with residues 130-169 of the apolipoprotein Emolecule in said mammal.

[0029] In one embodiment, pharmacological composition will comprise aglycosaminoglycan or derivative or fragment thereof along withpharmaceutically acceptable carriers, fillers or excipients. Theadministering step may comprise administering a pharmacologicalcomposition comprising an agent selected from the group consisting ofheparin, heparan sulfate, dermatan sulfate, dextran sulfate, pentosanpolysulfate, polyvinyl sulfate and fragments thereof along withpharmaceutically acceptable carriers, fillers or excipients. In a secondembodiment, the pharmacological composition will comprise a proteaseinhibitor, or a mixture of protease inhibitors along with one or morepharmaceutically acceptable carriers, fillers, or excipients. Theadministering step may comprise administering a pharmacologicalcomposition comprising a protease inhibitor “cocktail” such as a mixtureof aprotinin, leupeptin, pepstatin and antipain all available from SigmaChemical Co. Another embodiment will comprise a combination of the firstand second embodiments.

[0030] In another embodiment, pharmacological compositions will comprisecompounds containing napthaline sulfonic acids bonded to a phenyl ornaphthyl group by a diazo or amide bond thereof along withpharmaceutically acceptable carriers, fillers or excipients. Theadministering step may comprise administering an agent such as, but notlimited to, suramin sodium, ponceau S, Evan's blue, calconcarboxylicacid, Chicago sky blue 6b or mixtures thereof along withpharmaceutically acceptable carriers, fillers or excipients.

[0031] In a further embodiment, pharmacological compositions willcomprise compounds having a triphenylmethane core structure comprisingat least one carboxylate or sulfate substituent thereof along withpharmaceutically acceptable carriers, fillers or excipients. Thecarboxylate or sulfate substituents can be directly attached to thephenyl rings or can be part of a benzoic acid or benzene sulfonic acidattached to the triphenylmethane core. The administering step maycomprise administering an agent such as, but not limited to,aurintricarboxylic acid, aniline blue, Coomassie brilliant blue R-250,Coomassie brilliant blue G-250, light green SF yellowish, methyl blue ormixtures thereof along with pharmaceutically acceptable carriers,fillers or excipients.

[0032] In yet another embodiment, pharmacological compositions willcomprise compounds comprising tetrabromophenolsulfonpthalein along withpharmaceutically acceptable carriers, fillers or excipients. Theadministering step may compromise administering an agent such as, butnot limited to, bromophenol blue, bromocresol green or mixtures thereofalong with pharmaceutically acceptable carriers, fillers or excipients.

[0033] The method may be by oral administration of the interferingcompound or a pharmaceutically acceptable salt or derivative thereofinto said mammal.

[0034] The administering step comprises parenteral administration of thereceptor-binding site interfering compound or a pharmaceuticallyacceptable salt or derivative thereof into said mammal. Thisadministration may be by transdermal administration, subcutaneousinjection, intravenous injection, intraperitoneal injection,intramuscular injection, intrasternal injection, intrathecal injection,intraventricular and intracerebroventricular injection, inhalatoryspray, and infusion techniques.

[0035] The methods also comprise administering the receptor-binding siteinterfering compound or a pharmaceutically acceptable salt or derivativethereof along with a lipophilic solvent or carrier. The lipophilicsolvent or carrier may be an organic solvent, phosphatidyl choline andcholesterol.

[0036] The present method is useful in the treatment of a variety ofdiseases associated with apoE toxicity including, but not limited toAlzheimer's disease and Alzheimer-related senile dementia, cerebralamyloidosis, coronary heart disease, outcome following head injury orintracerebral hemorrhage, ischemic stroke, normal pressurehydrocephalus, ischemic cerebrovascular disease in end-stage renaldisease, glomerulopathy after renal transplantation, diabeticnephropathy, atherosclerosis, and HIV-associated dementia and peripheralneuropathy.

[0037] Accordingly, an object of the present invention is to providetreating a mammal having a condition associated with toxicity ofapolipoprotein E cleavage fragments containing residues 130-169,comprising administering to said mammal, in need of such treatment, apharmacologically effective amount of compound or a pharmaceuticallyacceptable salt, derivative or fragment thereof to interfere withgeneration of toxic fragments containing the receptor-binding siteassociated with residues 130-169 of the apolipoprotein E molecule insaid mammal or to interfere with the receptor binding site itself.

[0038] The present invention has several benefits and advantages. By useof the methods described, a safe and effective treatment may beadministered for a variety of diseases associated with apoE toxicitywithout the toxic side effects associated with many of the treatmentsavailable.

[0039] Other objects, features and advantages of the present inventionwill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and the specificexample, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the scope of the invention will become apparent tothose skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION

[0040] Apolipoprotein E (apoE), is a constituent of liver-synthesizedvery low density lipoproteins which function in the transport oftriglycerides from the liver to peripheral tissues. It is a recognitionsite for several cellular lipoprotein receptors, including hepatocytereceptors for chylomicron and VLDL remnants.

[0041] The term “apoE receptor-binding site (“ARS”), refers to thebinding site contained on the whole apoE protein and the 22 kDa fragmentand is responsible for binding of apoE with the low density lipoprotein(“LDL”) receptor or the LDL receptor-related protein (LRP) or otherrelated receptors. The apoE receptor binding site is associated withamino acid residues 130 through 169(Thr-Glu-Glu-Leu-Arg-Val-Arg-Leu-Ala-Ser-His-Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu-Arg-Asp-Ala-Asp-Asp-Leu-Gln-Lys-*Arg(Cys)-Leu-Ala-Val-Try-Gln-Ala-Gly-Ala-Arg-Glu-Gly; or asTEELRVRLASHLRKLRKRLLRDADDLQK-*R(C)-LAVYQAGAREG in single letter aminoacid shorthand) in the mature protein. The sequence shown occurs inisoforms E3 and E4. The E2 isoform is identical with the exception of acysteine substitution for arginine at position 158 denoted with anasterisk. The ARS overlaps the apoE heparin-binding site on the apoEprotein. The heparin-binding region is a positively charged,leucine-rich sequence, corresponding to amino acids 141-147(Leu-Arg-Lys-Leu-Arg-Lys-Arg; referred to as LRKLRKR in single letteramino acid shorthand) in the mature protein. ApoE peptides and proteinslacking these domains are not toxic to neurons.

[0042] The 22 kDa fragment of the apoE protein is the only major bandimmunoprecipitated from human brain and CSF samples by a monoclonalantibody that recognizes the putative toxic domain. This fragment runswith an apparent molecular weight of about 22 kDa, and likelycorresponds to the major apoE thrombin cleavage product, which has beenshown to be protease-resistant.

[0043] The term “biologically active” refers at least to the ability ofa proteinaceous molecule to specifically interfere with thereceptor-binding site of apoE (“ARS”), or to interfere with generationof toxic fragments of the apoE molecule having the apoE receptor-bindingsite, although other general or effector capability may be present inthat molecule as well.

[0044] Biological activity of an apoE receptor-binding interferingmolecule is evidenced by the interference with the binding of apoE withthe low density lipoprotein (“LDL”) receptor or the LDL receptor-relatedprotein or interference with the toxic effects of apoE peptidesincorporating ARS, at least at physiological pH values and ionicstrengths. Biological activity of a molecule interfering with generationof toxic fragments is widened by an ability to prevent generation oftoxic apoE fragments from full length apoE.

[0045] Preferably, biological activity occurs under biological assayconditions; i.e., those conditions within a pH value range of about 5 toabout 9, at ionic strengths such as that of distilled water to that ofabout one molar sodium chloride, and at temperatures of about 4° C. toabout 45° C.

[0046] “Derivative” refers to subject compounds having one or more aminoacid residues or carbohydrate moieties chemically derivatized byreaction of a functional group. Such derivatized molecules include, forexample, those molecules in which free amino groups have beenderivatized to form amine hydrochlorides, p-toluene sulfonyl groups,carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups orformyl groups. Free carboxyl groups may be derivatized to form salts,methyl and ethyl esters or other types of esters or hydrazides. Freehydroxyl groups may be derivatized to form O-acyl or O-alkylderivatives.

[0047] The term “fragment” refers to any subject compound having acomposition less than all of the molecule mentioned herein.

[0048] The term “glycosaminoglycan” is a general term for anymucopolysaccharide or sulfomucopolysaccharide. This is a general termfor a protein-polysaccharide complex obtained from proteoglycans andcontaining as much as 95% or more polysaccharide. All the known sixclasses of glycosaminoglycans contain amounts of glucosamine andgalactosamine. This class of compounds includes heparin, heparansulfate, dermatan sulfate and chondroitin sulfate A and C. Heparin is amucopolysaccharide comprised of D-glucuronic acid and D-glucosamine,both sulfated, in 1,4-α-linkage, of a molecular weight of from about6000 Da to about 20,000 Da. Heparan sulfate, or heparitin sulfate, is anheteropolysaccharide that has the same repeating disaccharide as heparinbut with fewer sulfates and more acetyl groups. Dermatan sulfate, orchondroitin sulfate B, is a mucopolysaccharide containing alternatingL-iduronic acid and N-acetyl-D-galactosamine 4-sulfate residues.Chondroitin is a mucopolysaccharide or proteoglycan composed ofalternating residues of β-D-glucuronic acid and N-acetylgalactosaminesulfate in alternating β1,3 and β1,4 linkages and are present in theground substance materials in the extracellular matrix of connectivetissue. Chondroitin sulfate A has sulfuric residues esterifying the4-hydroxyl groups of the galactosamine residues. Chondroitin sulfate Ccontains sulfuric residues esterifying the 6-hydroxyl groups of thegalactosamine residues.

[0049] The method of the present invention consists of treating a mammalhaving a condition associated with toxicity of apolipoprotein E cleavagefragments containing residues 130-169, comprising administering to saidmammal a pharmacologically effective amount of compound or apharmaceutically acceptable salt, derivative or fragment thereof tointerfere with the generation of toxic fragments of apoE or thereceptor-binding site and toxicity associated with residues 130-169 ofthe apolipoprotein E molecule in said mammal.

[0050] Amino acid residues 130-169 in human apoE encompass animmunoregulatory domain with both cytostatic and cytotoxic activitiesagainst interleukin-2 (‘IL2”)-dependent T cells. The positively charged,leucine-rich sequence, corresponding to amino acids 141-149(Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu; referred to as LRKLRKRLL in singleletter amino acid shorthand) in the mature protein represents theminimum sequence associated with the receptor-binding site (“ARS”), theheparin-binding site, and cytotoxicity. The present method utilizescompounds to interfere with the ARS. While not being bound by theory, itis believed that the interfering compounds will preferably associatetemporarily with the binding site or toxic fragment and, therefore,block access to the binding site to heparin. The association may be fromvery weak to very strong, depending on the compound utilized.Alternatively, a compound which forms a covalent bond may be utilized.

[0051] In one embodiment, the pharmacological composition willpreferably comprise a protease inhibitor or a glycosaminoglycan orderivative or fragment hereof along with pharmaceutically acceptablecarriers, fillers or excipients. Protease inhibitors, carriers, fillersand excipients are well known in the art. The administering step maycomprise administering a pharmacological composition comprising an agentselected from the group consisting of protease inhibitors, heparin,heparan sulfate, dermatan sulfate, dextran sulfate, pentosanpolysulfate, polyvinyl sulfate and fragments thereof along withpharmaceutically acceptable carriers, fillers or excipients.

[0052] In another embodiment, pharmacological compositions will comprisecompounds containing naphthalene sulfonic acids. The naphthalenesulfonic acids can be mono-, di-, or tri-napthalene sulfonic acids andmay comprise other substituents as well. Preferably, the naphthalenesulfonic acids will be covalently attached to a phenyl or naphthylmoiety. More preferably, the bond between the naphthalene sulfonic acidand the phenyl or naphthyl group will be a diazo or amide bond. Mostpreferably, two of the naphthalene sulfonic acid-phenyl molecules willbe bonded together through the phenyl groups to form larger, morecomplex molecules. Examples of such compounds are, but not limited to,ponceau S, direct blue 15, Evan's blue, amaranth, calconcarboxylic acid,suramin sodium, trypan blue, congo red, benzopurpurin 4b, Chicago skyblue 6b and sulfonazo III.

[0053] In a further embodiment, pharmacological compositions willcomprise compounds having a triphenylmethane core structure comprisingat least one carboxylate, sulfonate, benzoic acid or benzene sulfonicacid substituent. In a preferred embodiment, the compound will comprisecarboxylic acid substituents. More preferably , the compound willcomprise a carboxylic acid substituent on each phenyl moiety oftriphenylmethane and most preferably, the compound will beaurintricarboxylic acid. In another preferred embodiment, the phenylrings of triphenylmethane will comprise at least one benzene sulfonicacid substituent. More preferably, the benzene sulfonic acid will becovalently attached to one of the phenyl rings through a nitrogen bond.Examples of such compounds are, but not limited to, aniline blue, methylblue, Coomassie brilliant blue R-250, Coomassie brilliant blue G-250 andmixture thereof.

[0054] In yet another embodiment, pharmacological compositions willcomprise compounds comprising tetrabromophenolsulfonphthalein. Examplesof such compounds are, but not limited to, bromophenol blue andbromocresol green.

[0055] In another embodiment, pharmacological compositions will comprisecompounds chosen from the group consisting essentially of cibacron blue,thiazol yellow G, sulfobromophthalein, biebrich scarlet, or mixturesthereof.

[0056] It will be appreciated that pharmacological compositions of thepresent invention can comprise a mixture of any of the compounds of thepresent invention along with pharmaceutically acceptable carriers,fillers or excipients.

[0057] The methods may be by oral administration of the interferingcomposition or a pharmaceutically acceptable salt or derivative thereofinto said mammal. The methods according to the present inventionpreferably allows the administration of the interfering molecule in aunitary dose of from about 1 to about 1000 mg. A unitary dose isgenerally administered from about 1 to about 3 times a day.

[0058] The administering step may comprise parenteral administration ofthe receptor-binding site interfering compound or a pharmaceuticallyacceptable salt or derivative thereof into said mammal. Thisadministration may be by transdermal administration, subcutaneousinjection, intravenous injection, intraperitoneal injection,intramuscular injection, intrasternal injection, intrathecal injection,intracerebroventricular injection, inhalatory spray and infusiontechniques.

[0059] The method also comprises administering interfering compound or apharmaceutically acceptable salt or derivative thereof along with alipophilic compound, such as a lipophilic solvent or carrier. Thelipophilic solvent or carrier may be an organic solvent, phosphatidylcholine and cholesterol.

[0060] The present method is useful in the treatment of a variety ofdiseases associated with apoE toxicity including, but not limited toAlzheimer's disease and Alzheimer-related senile dementia, cerebralamyloidosis, coronary heart disease, outcome following head injury orintracerebral hemorrhage, ischemic stroke, normal pressurehydrocephalus, ischemic cerebrovascular disease in end-stage renaldisease, glomerulopathy after renal transplantation, diabeticnephropathy, atherosclerosis, and HIV-associated dementia and peripheralneuropathy. Suitable subjects may include those diagnosed as afflictedwith Alzheimer's disease or any of the other conditions cited above. Thepresent invention may be employed in treating both familial Alzheimer'sdisease (late onset and early onset) as well as sporadic Alzheimer'sdisease. Many Alzheimer's disease patients encountered in practice haveno obvious family history and have been classified as sporadic. However,genetic factors in early and late-onset of familial Alzheimer's disease(FAD) are well documented. Late-onset Alzheimer's disease is theclassification usually used if the disease is diagnosed to occur afterthe age of 65 in humans.

[0061] It is preferred and contemplated that the methods describedherein be used in conjunction with clinical diagnostic information knownor described in the art which are used in evaluation of subjects withAlzheimer's disease or suspected to be at risk for developing suchdisease or other apoE-related diseases.

[0062] Studies have shown that apoE fragments ranging from 5 to 22 kDaare present in the post-mortem cerebral spinal fluid from both controlpatients and patients with AD. The only major band immunoprecipitated bya monoclonal antibody that recognizes the putative toxic domain runswith an apparent molecular weight of about 22 kDa. This fragment likelycorresponds to the major apoE thrombin cleavage product. Interferencewith the production of apoE fragments provides an alternative treatmentstrategy.

[0063] Purified apoE, derived from transfected HEK cells, subjected tothrombin cleavage and separated using gel filtration to collect the 22kDa fragment, yields enhanced toxicity when tested against primaryneurons in culture. The 22 kDa fragments purified from the E4 isoform ismore toxic than E3-derived fragments. The putative toxic site is closelyassociated with one of two well-characterized heparin binding domainsassociated with residues 141-147 of apoE.

[0064] The density of four positively charged amino acid residues in the141-149 domain clearly make a significant contribution to apoE peptidetoxicity. Consistent with this conclusion is the ablation ofpeptide-mediated toxicity by the polyanionic glycosaminoglycans (“GAG”)heparin, heparan sulfate and chondroitin sulfate. Furthermore, not allGAGs show inhibition of toxicity.

[0065] In one embodiment, the site-interfering compound may be aglycosaminoglycan or derivative or fragment thereof. It is known thatglycosaminoglycans are products capable of being obtained by extractionfrom animal tissues. Certain of these glycosaminoglycans have veryadvantageous anticoagulating and antithrombotic properties. Typicalproducts of this family are heparin, its cleavage products and theirderivatives, as well as heparan sulfate and dermatan sulfate. However,preferred methods of the present invention will utilize compoundswithout potent anticoagulant properties.

[0066] In particular, it is known that dermatan sulfate is a family ofpolymers with a variable degree of polymerization, formed of repeatingunits consisting of a uronic acid group (iduronyl or glucuronyl) and ofan acetyl 4-sulphated galactosaminyl group (H. W. Stuhlsatz, “TheMethodology of Connective Tissue Research”, (1976), 137-146). Naturaldermatan sulfate has a molecular mass of between 2×10⁴ and 4×10⁴D. Thisproduct is particularly advantageous as an anticoagulant andantithrombin (F. Fernandez et al., British Journal of Haematology,(1986), 64, 309-317). The heparin-binding site interfering compound maybe a glycosaminoglycan or derivative or fragment thereof with amolecular mass of between 2×10¹ and 4×10⁵D.

[0067] It is known that the main heparin chain is constructed in twostages. In a first stage, heparin is biosynthesized from a precursorproteoglycan whose polysaccharide part consists of a family of polymerswith a variable degree of polymerization formed from repeating beta-D-glucuronyl-1,4-alpha-N-acetyl-D-glucosaminyl-(1,4) disaccharideunits. This polysaccharide part is generally called N-acetylheparosan(Navia, J., Anal. Biochem. 135:134-140 (1983)). This first stage ofbiosynthesis is the only time when it is truly possible to speak of a“disaccharide unit” because the second stage of the biosynthesis willprofoundly change this simple skeleton (“L'heparin, fabrication,structure, properties, analyses”, J. P. Duelos, (1984) pp. 81-83, MassonEd.-France).

[0068] Natural heparin resulting from biosynthesis is a polysaccharideconsisting of molecules of glucuronic acid and of iduronic acid (uronicacids), optionally sulphated in position 2, combined with molecules ofglucosamine, optionally sulphated in position 6 and sulphated oracetylated on the amine in position 2. The heparin-binding siteinterfering compound may be a compound derived from natural heparin.

[0069] The natural heparin used as the starting material can be standardheparin or any other commercially available heparin, provided that ithas a good quality. A sodium salt of heparin may be used, even if othersalts can conveniently be used. It is preferable that starting heparinbe anhydrous, hence a preliminary dehydration is properly performed, forexample, at a temperature of from about 50°to about 60° C. Theinterfering compound may be derived from natural heparin throughcleavage with enzymes or through other chemical means.

[0070] In one embodiment, the ARS blocking compound may be, for example,dermatan sulfate (#03125 and #03120), heparan sulfate 1 (#03100),heparan sulfate 2 (#03400), LP heparin fraction (#03010), and HP heparinfraction (#03020) (all from Celsus Laboratories, Inc., Cincinnati,Ohio). Other compounds include heparin sulfate (Product Nos. H5393,H9902, H7640, H9637, and H7641) and heparin (Product Nos. H0880, H8398,and H0878) (all from Sigma Chemical Co., St. Louis, Mo.).

[0071] In another embodiment, the ARS blocking compound may comprisecompounds containing naphthalene sulfonic acids. The naphthalenesulfonic acids can be mono-, di-, or tri-napthalene sulfonic acids andmay comprise other substituents as well. Preferably, the naphthalenesulfonic acids will be covalently attached to a phenyl or naphthylmoiety. More preferably, the bond between the naphthalene sulfonic acidand the phenyl or naphthyl group will be a diazo or amide bond. Mostpreferably, two of the naphthalene sulfonic acid-phenyl molecules willbe bonded together through the phenyl groups to form larger, morecomplex molecules. Examples of such compounds are, but not limited to,ponceau S, direct blue 15, Evan's blue, amaranth, calconcarboxylic acid,suramin sodium, trypan blue, congo red, benzopurpurin 4b, Chicago skyblue 6b and sulfonazo III.

[0072] In a further embodiment, the ARS blocking compound may comprisecompounds having a triphenylmethane core structure comprising at leastone carboxylate, sulfonate, benzoic acid or benzene sulfonic acidsubstituent. In a preferred embodiment, the compound will comprisecarboxylic acid substituents. More preferably, the compound willcomprise a carboxylic acid substituent on each phenyl moiety oftriphenylmethane and most preferably, the compound will beaurintricarboxylic acid. In another preferred embodiment, the phenylrings of triphenylmethane will comprise at least one benzene sulfonicacid substituent. More preferably, the benzene sulfonic acid will becovalently attached to one of the phenyl rings through a nitrogen bond.Examples of such compounds are, but not limited to, aniline blue, methylblue, Coomassie brilliant blue R-250, Coomassie brilliant blue G-250 andmixture thereof.

[0073] In yet another embodiment, the ARS blocking compound may comprisecompounds comprising tetrabromophenolsulfonphthalein. Examples of suchcompounds are, but not limited to, bromophenol blue and bromocresolgreen.

[0074] In another embodiment, the ARS blocking compound may comprisecompounds chosen from the group consisting essentially of cibacron blue,thiazol yellow G, sulfobromophthalein, biebrich scarlet, or mixturesthereof.

[0075] It will be appreciated that the ARS blocking compound of thepresent invention may comprise a mixture of any of the compounds of thepresent invention along with pharmaceutically acceptable carriers,fillers or excipients.

[0076] The pharmaceutical compositions of the present invention can beformulated for oral, sublingual, subcutaneous, intravenous, transdermicor rectal administrations in dosage units and in admixture withpharmaceutical excipients or vehicles. Convenient dosage forms include,among those for oral administration, tablets, powders, granulates, and,among those for parenteral administration, solutions especially fortransdermal administration, subcutaneous injection, intravenousinjection, intraperitoneal injection, intramuscular injection,intrasternal injection, intrathecal injection, inhalatory spray, andinfusion techniques.

[0077] The pharmaceutical compositions of the present invention areadministered, in the above mentioned forms and routes, to animals andman in case of a pathological increase in toxicity of apoE fragmentscontaining the residue sequence 130-169, particularly in the treatmentof Alzheimer's disease.

[0078] The daily amount in the aforesaid indications may range from 0.1to 100 mg/kg and each unitary dose may contain from 1 to 1000 mg of theactive ingredient. Such unitary dose can be administered from 1 to 3times a day for the treatment of dementia disorders and atherosclerosis.

[0079] The dosage can vary widely as a function of the age, weight andstate of health of the patient, the nature and the severity of theailment, as well as of the administration route. These doses cannaturally be adjusted for each patient according to the results observedand the blood analyses previously carried out.

[0080] The following examples illustrate and explain the presentinvention but should not be taken as limiting the present invention inany regard.

EXAMPLE 1

[0081] Since the 22 kDa fragment contains the domain associated withtoxicity and corresponds to the major N-terminal proteolytic fragment ofapoE, assessment of the efficacy of a compound can first be screened bymeasuring the inhibition of the 22 kDa toxicity in vitro. The 22 kDathrombolytic cleavage fragment product of apoE is neurotoxic and ispurified from medium of HEK cells transfected with the gene for humanapoE4. Neuronal toxicity is then assessed using dissociated embryonicchick sympathetic neurons in 96 well microtiter plates. Following theaddition of the fragments, the cultures are incubated overnight.Viability is then assessed by vital dye staining.

EXAMPLE 2 Effects of apoE 22 kDa Peptides on Sympathetic Neurons inCulture

[0082] Transfected HEK cells are cultured as previously described (LaDu,M. J., et al., J. Biol. Chem. 258:12348-54 (1983)). The apoE isconcentrated from conditioned medium by ultrafiltration (10 kDa cut-offmembrane, Amicon) followed by heparin column chromatography(heparin-coupled agarose beads, Sigma). The purified apoE is thendigested with thrombin and the resulting fragments are separated by HPLCgel filtration chromatography (Bio SEC-Bio Rad). After buffer exchangewith centricon 10 (Amicon) and lyophilization, the purified 22 kDafragment is subjected to amino acid analysis. For neurotoxicity studies,lumbar sympathetic chain ganglia are isolated from embryonic day ninechicken embryos (Spafas, Inc., Roanoke, Ill.) under sterile conditionsin unsupplemented Ham's F12 medium (Sigma). The dissected chains areexposed to trypsin (0.25%) for 20 minutes at 37° C. The trypsin isinactivated by adding fetal bovine serum. The chains are washed threetimes with medium and then triturated with flamed Pasteur pipettes todissociate the cells. The cells are resuspended in Neurobasal medium(Gibco) and plated into 96-well plates pretreated with poly-ornithine.Dishes are incubated at 37° C. with 5% CO₂/95% air. Following overnightincubation, the cells are treated with dermatan sulfate (#03125, CelsusLaboratories, Inc., Cincinnati, Ohio), diluted in F12 mediumsupplemented with 100 μM putrescine, 20 nM progesterone, 100 μg/ml humantransferrin, 30 nM selenium and 1% antibiotics(penicillin-streptomycin). The average molecular weight is approximately35 kDa. The neuronal cells are pre-incubated for 10 min. at 37° C. with4 μM of the dermatan sulfate. The toxic fragments are added to theculture medium. Controls use the corresponding vehicle. After overnightincubation, the cells are labeled with a vital dye (5-carboxyfluoresceindiacetate, acetoxymethyl ester, Molecular Probes, Eugene, Oreg.) for 30minutes. The wells are washed with fresh F12 medium and images of thestained cells are collected from each well by using a Diaphot invertedfluorescence microscope connected to a Macintosh IIfx computer equippedwith a Framegrabber video card. The number of labeled neurons isquantified from the stored images with NIH Image software (version1.57). The results show the number of cells surviving followingovernight exposure to different concentrations of either the E3- orE4-derived 22 kDa fragment. Those treated with the dermatan sulfate showtoxicity is completely ablated.

EXAMPLE 3

[0083] Same as above only neuronal toxicity is tested with heparansulfate (#03105, Celsus Laboratories, Inc., Cincinnati, Ohio).

EXAMPLE 4

[0084] Same as above only neuronal toxicity is tested with the highpotency heparin fraction (#03025, Celsus Laboratories, Inc., Cincinnati,Ohio).

EXAMPLE 5

[0085] Results of in vitro experiments showing interference effect of aprotease inhibitor mixture.

[0086] Full-length apoE4 (8 μM) was added to primary chick sympatheticneurons in cultures using the procedure of Example 1 in the presence andabsence of a mixture of protease inhibitors obtained from Sigma ChemicalCo.: protease inhibitor concentration aprotinin 1.5 ± 0.5 μg/mlleupeptin 1.5 ± 0.5 μg/ml pepstatin A 1.5 ± 0.5 μg/ml antipain 1.5 ± 0.5μg/ml

[0087] The protease inhibitor mixture was found to significantly reducethe toxicity of the apoE and to reduce the production of apoE fragments(based on Western blotting). The same protease inhibition mixture didnot block the toxicity of the apoE fragment or the long tandem apoEpeptide. Therefore, the method of interfering is likely not due tointerfering with the binding of apoE fragments to the receptor, but dueto prevention of generation of toxic fragments.

EXAMPLE 6 Protection of Sympathetic Neurons Against Toxic apoE Fragmentsby Various Compounds.

[0088] Transfected HEK cells were cultured as previously described(LaDu, M. J., et el., J. Biol. Chem.258:12348-54 (1983).) The apoE wasconcentrated from conditioned medium by ultrafiltration (10 kDa cut-offmembrane, Amicon) followed by heparin column chromatography(heparin-coupled agarose beads, Sigma). The purified apoE was thendigested with thrombin and the resulting fragments separated by HPLC gelfiltration chromatography (Bio SEC-Bio Rad). After buffer exchange andlyophilization, the purified 22 kDa fragment was subjected to amino acidanalysis. For neurotoxicity studies, lumbar sympathetic chain gangliawere isolated from embryonic day nine chicken embryos (Spafas, Inc.,Roanoke, Ill.) under sterile conditions in unsupplemented Ham's F12medium (Sigma). The dissected chains were exposed to trypsin (0.25%) for20 minutes at 37° C. The trypsin was inactivated by addition of fetalbovine serum. The chains were washed three times with medium and thentriturated with flamed Pasteur pipettes to dissociate the cells. Thecells were resuspended in Neurobasal medium (Gibco) and plated into96-well plates pretreated with poly-ornithine. Dishes were incubated at37° C. with 5% CO₂/95% air. Following overnight incubation, the cellswere treated with a range of concentrations of various compounds dilutedin F12 medium supplemented with 100 μM putrescine, 20 nM progesterone,100 μg/ml human transferrin, 30 nM selenium and 1% antibiotics(penicillin-streptomycin). The neuronal cells were pre-incubated for 10min at 37° C. with a predetermined concentration of compound. The toxicfragments were then added to the culture medium while controls used thecorresponding vehicle without the toxic fragments. After overnightincubation, the cells were labeled with a vital dye(5-carboxyfluorescein diacetate, acetoxymethyl ester, Molecular Probes,Eugene, Oreg.) for 30 minutes. The wells were washed with fresh F12medium and images of the stained cells were collected from each wellusing a Diaphot inverted fluorescence microscope connected to aMacIntosh IIfx computer equipped with a Framegrabber video card. Thenumber of labeled neurons was quantified from the stored images with NIHImage software (version 1.57). The results shown in Table 1 areexpressed as C₅₀, the concentration required to prevent 50% cell deathas compared to control. TABLE 1 Compound C₅₀ (μM) potassium polyvinylsulfate 0.035 pentosan sulfate 1.5 dextran sulfate (5 kDa) 0.8 heparansulfate 1.0 ponceau S 5 direct blue 15 18 Evan's blue 8 amaranth 25calconcarboxylic acid 10 suramin sodium 5 trypan blue 20 congo red 25benzopurpurin 4b 50 Chicago sky blue 6b 15 sulfonazo III 45aurintricarboxylic acid 11 aniline blue 25 methyl blue 20 Coomassiebrilliant blue G-250 40 Coomassie brilliant blue R-250 35 light greenS.F. yellowish 40 bromophenol blue 16 bromocresol green 25 cibacron blue20 thiazol yellow G 25 sulfobromophthalein 32 biebrich scarlet 40

[0089] The foregoing discussion discloses and describes merely exemplaryembodiments of the present invention. One skilled in the art willreadily recognize from such discussion, and from the accompanyingclaims, that various changes, modifications and variations can be madetherein without departing from the spirit and scope of the invention asdefined in the following claims.

[0090] All references cited herein are incorporated by reference as iffully set forth herein.

What is claimed is:
 1. A method of preventing apolipoprotein E toxicityto a cell comprising treating said cell with a compound capable ofinhibiting apolipoprotein E toxicity.
 2. The method of claim 1, whereinthe compound is selected from the group consisting of polyvinyl sulfate,pentosan polysulfate, dextran sulfate, heparan sulfate and mixturesthereof.
 3. The method of claim 1, wherein the compound furthercomprises napthalenesulfonic acid covalently bonded to a phenyl ornaphthyl group.
 4. The method of claim 2, wherein the napthalenesulfonicacid is covalently bonded to a phenyl or naphthyl group through a diazoor amide bond.
 5. The method of claim 1, wherein the compound isselected from the group consisting of ponceau S, Evan's blue, suraminsodium, direct blue 15, calconcarboxylic acid, amaranth, trypan blue,congo red, benzopurpurin 4b, Chicago sky blue 6b, sulfonazo III andmixtures thereof.
 6. The method of claim 1, wherein the compound furthercomprises a triphenylmethane core modified with at least one sulfate orcarboxylate group.
 7. The method of claim 1, wherein the compoundfurther comprises a triphenylmethane core modified with at least onebenzoic acid or benzenesulfonic acid substituent.
 8. The method of claim7, wherein the benzoic acid or benzenesulfonic acid substituents arecovalently bound to the phenyl groups of the triphenylmethane through anitrogen bond.
 9. The method of claim 1, wherein the compounds areselected from the group consisting of aurintricarboxylic acid, anilineblue, methyl blue, light green SF yellowish, Coomassie brilliant blueG-250, Coomassie brilliant blue R-250, and mixtures thereof.
 10. Themethod of claim 1, wherein the compound further comprises atetrabromophenolsulfonphthalein.
 11. The method of claim 1, wherein thecompound is selected from the group consisting of bromophenol blue,bromocresol green and mixtures thereof.
 12. The method of claim 1,wherein the compound is selected from the group consisting of cibacronblue, thiazol yellow G, sulfobromophthalein, biebrich scarlet andmixtures thereof.
 13. The method of claim 1, wherein inhibitingapolipoprotein E toxicity comprises inhibiting binding of apolipoproteinE or fragments of apolipoprotein E to a cell.
 14. The method of claim 1,wherein the fragments of apolipoprotein E comprise residues 141-147 ofapolipoprotein E.
 15. The method of claim 1, wherein inhibitingapolipoprotein E toxicity comprises inhibiting production of a peptidefragment of apolipoprotein E comprising residues 141-147 ofapolipoprotein E.
 16. A method of treating conditions associated withapolipoprotein E toxicity, comprising administering a compositioncomprising a pharmacologically effective amount of a compound thatinhibits apolipoprotein E toxicity.
 17. The method of claim 16, whereinthe compound is selected from the group consisting of polyvinyl sulfate,pentosan polysulfate, dextran sulfate, heparan sulfate and mixturesthereof.
 18. The method of claim 16, wherein the compound furthercomprises napthalenesulfonic acid covalently bonded to a phenyl ornaphthyl group.
 19. The method of claim 18, wherein thenapthalenesulfonic acid is covalently bonded to a phenyl or naphthylgroup through a diazo or amide bond.
 20. The method of claim 16, whereinthe compound is selected from the group consisting of ponceau S, Evan'sblue, suramin sulfate, direct blue 15, calconcarboxylic acid, amaranth,trypan blue, congo red, benzopurpurin 4b, Chicago sky blue 6b, sulfonazoIII and mixtures thereof.
 21. The method of claim 16, wherein thecompound further comprises a triphenylmethane core modified with atleast one sulfate or carboxylate group.
 22. The method of claim 16,wherein the compound further comprises a triphenylmethane core modifiedwith at least one benzoic acid or benzenesulfonic acid substituent. 23.The method of claim 22, wherein the benzoic acid or benzenesulfonic acidsubstituents are covalently bound to the phenyl groups of thetriphenylmethane through a nitrogen bond.
 24. The method of claim 16,wherein the compounds are selected from the group consisting ofaurintricarboxylic acid, aniline blue, methyl blue, light green SFyellowish, Coomassie brilliant blue G-250, Coomassie brilliant blueR-250, and mixtures thereof.
 25. The method of claim 16, wherein thecompound further comprises a tetrabromophenolsulfonphthalein.
 26. Themethod of claim 16, wherein the compound is selected from the groupconsisting of bromophenol blue, bromocresol green and mixtures thereof.27. The method of claim 16, wherein the compound is selected from thegroup consisting of cibacron blue, thiazol yellow G,sulfobromophthalein, biebrich scarlet and mixtures thereof.
 28. Themethod of claim 16, wherein inhibiting apolipoprotein E toxicitycomprises inhibiting binding of apolipoprotein E or fragments ofapolipoprotein E to a cell.
 29. The method of claim 28, wherein thefragments of apolipoprotein E comprise residues 141-147 ofapolipoprotein E.
 30. The method of claim 16, wherein inhibitingapolipoprotein E toxicity comprises inhibiting production of a peptidefragment of apolipoprotein E comprising residues 141-147 ofapolipoprotein E.
 31. The method of claim 16, wherein the condition isAlzheimer's-type senile dementia.
 32. The method of claim 16, whereinthe condition is a condition associated with cerebral amyloidosis. 33.The method of claim 16, wherein the condition is hyperlipidemia.
 34. Themethod of claim 16, wherein the condition is selected from the groupconsisting of coronary heart disease, atherosclerosis, head injury,ischemic stroke, intracerebral hemorrhage, normal pressurehydrocephalus, HIV-associated dementia and HIV-associated peripheralneuropathy.